Nucleolin-mediated cancer diagnostics and therapy

ABSTRACT

The present invention provides for diagnostic kits for identifying cancer patients who are more susceptible to cancer therapies employing endostatin and other angiogenesis inhibitors, based upon the discovery that Nucleolin is a specific receptor for Endostatin. In particular, the diagnostic kits include antibody molecules against Nucleolin, DNA or RNA molecules that specifically bind to nucleic acid molecules encoding Nucleolin. The present invention also discloses methods of screening for angiogenesis inhibitors which specifically interact with Nucleolin, and act as angiogenesis inhibitors in an analogous manner as Endostatin. In addition, the present invention discloses methods of inhibiting the proliferation of endothelial cells or angiogenesis of tumor by administering an anti-nucleolin antibody linked to a cytotoxic agent such as tumor necrosis factor alpha to the endothelial cells.

RELATED APPLICATION

This application claims priority to Chinese Patent Application, SerialNo. 200510011707.3, filed May 12, 2005, the entire content of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel method of identifying cancersubjects, in particular human patients, who are suitable candidates foranti-angiogenesis cancer therapy. The present invention also relates toa novel approach in searching and screening for angiogenesis inhibitors,molecules which are believed to be effective in reducing the malignantgrowth of cells, particularly in those cancers which areangiogenesis-dependent. The present invention discloses methods ofscreening for inhibitors of angiogenesis using the molecule nucleolin.In particular, the present invention relates to screening forangiogenesis inhibitors which functions in a manner that is analogous tothe protein endostatin. The invention is based upon the discovery thatnucleolin is a specific receptor for endostatin, and is involved in thesignal transduction pathway of endostatin when it functions as anangiogenesis inhibitor.

2. Description of Related Art

The effectiveness of cancer therapy can vary greatly among targetedpatients depending upon a variety of factors, both external andinternal. External factors include the different stages of the cancer atthe time of treatment, where early detection is key to effectivetreatment and recovery, the relevant strength of the cancer therapy,such as surgery, chemotherapy, or radiation therapy. Internal factorsinclude the health of the immune system of the patient, where a strongsystem can sustain a longer and stronger regiment of treatment, and thushelping the patient recover faster. A key issue that is being exploredin the cancer therapy, and in medicine in general, in what's calledpersonalized medicine. The notion that different individuals may havedifferent tolerance and susceptibility to the same cancer drug ortherapy has inspired a variety of approaches in the effort to increasethe efficacy of a particular cancer therapy. Thus, due to individualvariations, a drug that is effective on one patient may not be so onanother.

In the field of cancer therapy, attempts have been made to use geneprofiling to understand whether a particular drug can exert itstherapeutic function effectively on a patient with a particular geneticprofiling. One approach of cancer therapy discovered recently has beenusing endostatin to inhibit the angiogenesis of tumor cells, and thusinhibiting the growth of tumor by stopping blood supply to the tumors.To understand the individualized application of endostatin therapy,recently, there have been attempts to use gene microarray to study thegene expression profiling underlying the inhibitory effects ofendostatin on angiogenesis of endothelial cells. See M. Mazzanti, etal., Genome Research, 14:1585-1593 (2004).

In the context of endostatin cancer therapy, endostatin has been hailedas an effective cancer therapy because it works to kill cancer cells byinhibiting angiogenesis, a process which is required by the cancer cellsin order to metastasize. Every increase in the tumor cell populationmust be preceded by an increase in new capillaries that converge uponthe tumor. This phenomenon is nearly universal; most of the human solidtumors and hematopoietic malignancies are angiogenesis dependent.Additional advantages to antiangiogenic therapy include low toxicity,minimal drug resistance, and repeated cycles of antiangiogenic therapymay be followed by a prolonged tumor dormancy without further therapy.See Boehm et al., Antiangiogenic therapy of experimental cancer does notinduce acquired drug resistance. Nature (1997) 390: 404-407. However, sofar the mechanism of function of how endostatin works is still notclear. Therefore, endostatin therapy is being applied to cancer patientsacross the board without referencing to each patient's susceptibility ofthe therapy. Numerous clinical trials have been conducted in hope offinding an effective drug for anti-angiogenesis. It would be asignificant progress if patients can be selected using objectivecriteria so they can be treated more effectively with endostatin. Thisinvention discloses methods and diagnostic kits directed to thisendeavor.

SUMMARY OF INVENTION

The present invention provides a kit for determining the susceptibilityof a subject to endostatin cancer therapy, comprising a label thatlabels nucleolin and a usage instruction for performing a screening of asample of said subject with said label such as that an amount ofnucleolin present in the sample is determined. Preferably, the subjectis a mammal. More preferably, the subject is a human. In certainpreferred embodiment of the invention, the nucleolin being labeled iscell surface nucleolin. In other embodiment of the invention, the labelcomprises an antibody that specifically binds to nucleolin, preferably apolyclonal antibody, and more preferably, a monoclonal antibody. In yetanother embodiment of the invention, the label comprises a nucleic acidmolecule, or probe, preferably a DNA probe, and still preferably, an RNAprobe.

The present invention further provides a method of determining thelikelihood of success of endostatin cancer therapy in a subject,comprising screening a sample from said subject for the level ofexpression of nucleolin, and determining if said subject is susceptibleto endostatin cancer therapy based on the amount of nucleolinexpression.

The present invention provides methods of screening for angiogenesisinhibitors, in particular, molecules which function in a similarlymanner as endostatin. The invention provides methods of using Nucleolin,referred to herein as “NL”, as a target molecule, applying conventionalmethodologies, candidate molecules can be examined so as to uncoverthose molecules that bind specifically to NL, at the same time,exhibiting angiogenesis activities. Due to the fact that NL is aspecific cell surface receptor for endostatin, the new moleculesdiscovered in the manner described above should function in a similarlymanner as endostatin.

The present invention also provides methods of enhancing the sensitivityof target endothelial cells to endostatin. The methods herein provideintroducing exogenous NL molecules into the target endothelial cells,such that the NL molecules are over-expressed as compared to their wildtype levels. Preferably, these target cells are those that normally donot express a high level NL endogenously. The methods further providethe introduction of NL to targeted endothelial cells, such that thesemodified cells can be effectively killed by endostatin due toendostatin's angiogenesis properties. The present invention alsoprovides for antibodies against NL molecules, which can be used todetect target cancer cells having a high level of surface NL and as suchare good candidate for ES cancer therapy.

In one embodiment, the present invention provides a method of producingan NL-specific angiogenesis inhibitor effective in inhibitingangiogenesis, comprising: applying an appropriate binding assay to apool of candidate molecules, thereby obtaining a plurality ofNL-specific molecules; testing each of the plurality of NL-specificmolecules for its effectiveness of inhibiting angiogenesis using ananti-angiogenesis assay; and selecting the resulting NL-specificmolecule which is effective in inhibiting angiogenesis as demonstratedby the anti-angiogenesis assay.

In another embodiment, the present invention provides a method ofselecting an angiogenesis inhibitor having the ability to inhibitendothelial proliferation when added to proliferating endothelial cellsin vitro, comprising the steps of: using a pharmaceutically acceptablemethod to discover molecules that specifically interact with NL as thetarget molecule; testing the molecules thus derived from the previousstep for their effectiveness in inhibiting endothelial cellproliferation or migration; and harvesting the molecule thus derivedwhich are effective in inhibition of endothelial cell proliferation ormigration, wherein the effectiveness of the anti-angiogenesis functionof said molecule is compared to that of Endostatin.

In a further embodiment, the present invention provides a method ofincreasing the receptiveness of a target cell to an angiogenesisinhibitor, comprising: introducing exogenous NL into the target cells,thereby obtaining a plurality of modified target cells expressingexogenous NL, and measuring the killing rate of the modified targetcells by endostatin.

In another embodiment, the present invention provides a method ofenhancing the anti-angiogenesis effect of an angiogenesis inhibitor on atarget endothelial cell, comprising introducing into said target cell apharmaceutically effective amount of exogenous NL molecule, said NLmolecule being able to express in said target cell; and incubating saidtarget cell with said angiogenesis inhibitor, thereby causing theinhibition of the growth of said target cell.

In one embodiment, the present invention provides a method of increasingthe efficacy of angiogenesis inhibitors on controlling the growth of acancer in a patient having such cancer, comprising identifying thepresence of the level of endogenous NL molecules in a sample of thecancer of said patient, using acceptable methods; and determining thelikelihood of efficacy of angiogenesis inhibitor on said cancer patientusing the level of expression of NL in said patient, a higher level ofNL indicating a higher degree of success of such angiogenesis inhibitortreatment. Preferably, the angiogenesis inhibitor is endostatin.

In another embodiment, the present invention provides a diagnostic kitfor assaying the individual sensitivity of target cells towardsangiogenesis inhibitors, comprising a molecule that specifically bind toan NL molecule; and a pharmaceutically acceptable carrier. Preferably,the molecule is a polyclonal antibody, and more preferably, the antibodyis a monoclonal antibody.

In a further embodiment, the present invention provides a method ofidentifying target cancer cells which are susceptible to ananti-angiogenesis inhibitor treatment, comprising generating ananti-nucleolin antibody; using said anti-nucleolin antibody to screen asample of said target cancer cells; and identifying target cancer cellssusceptible to anti-angiogenesis inhibitor treatment as indicated by thespecific interactions between the target cancer cells and theanti-nucleolin antibody. Preferably, the antibody is a polyclonalantibody, and more preferably, the antibody is a monoclonal antibody.

In yet another embodiment, the present invention provides a diagnostickit for determining the target cancer cells which are susceptible toanti-angiogenesis inhibitor treatment, comprising an antibody againstnucleolin and a pharmaceutically acceptable carrier.

In yet another embodiment, the present invention provides a diagnostickit for determining and selecting cancer subjects which are susceptibleto anti-angiogenesis inhibitor treatment, comprising a label that iscapable of binding to nucleolin and indicating its presence, and awritten instruction for performing a screening test of a sample of thecancer subject such that the level of presence of nucleolin is detectedin the sample.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows that HMEC (human microvascular endothelial cells) is asensitive cell line in response to ES in migration and proliferation. a,Cell migration assay were performed with HMECs in the presence of ES atconcentrations as indicated, PBS serves as a control. b, Cellproliferation assay of HMECs was determined in the presence of ES atconcentrations as indicated, and PBS serves as a control. The number ofcells was evaluated by MTT assay. Results are means±s.e.m., n=3 (a), andn=5 (b).

FIG. 2 shows that ES binds to cell surface NL. a, ES binding proteinsisolated from the cell surface of HMECs were identified as NL and itsfragment. The ES binding proteins were isolated from HMECs' plasmamembrane using ES-Ni-NTA affinity beads as described in methods. Thefractions eluted with 500 mM sodium chloride in PBS buffer were appliedto SDS-PAGE (left panel) and immunoblotting using monoclonal antibodiesagainst NL (right panel). b, ES binds NL specifically in vitro.Immunoprecipitation was carried out using recombinant NL and ES. c,Heparin interrupts the formation of ES-NL complex. Immunoprecipitationusing recombinant NL and ES was carried out in vitro with or withoutheparin (200 nM). d, ES binds HMECs specifically via its surface NL.HMECs were incubated with ES and different concentrations of antibodiesagainst NL for 30 min at RT, and then were washed for 3 times with PBSbuffer. The cells were applied to SDS-PAGE and immunoblotting usingantibodies against ES. β-actin was used as control. C, HMECs wereincubated with ES (20 μg/ml) for different periods of time at 37° C. and5% CO₂. After washed with fresh medium, immunoprecipitation andimmunoblotting was carried out with antibodies against NL and ES,respectively. β-actin was used as control. f-i, co-localization of NLand ES on the surface of HMEC cells. Intact HEMC cells were stained withboth mouse anti-NL antibody, and rabbit anti-ES antibody. Examinationwas conducted by indirect immunofluorescence using laser scanningconfocal microscopy. Scale bar, 20 μm.

FIG. 3 shows that nucleolin is a receptor for endostatin. a, Indicatedconcentrations of recombinant NL were tested to lift the inhibition ofES in HUVECs migration assay, PBS serves as a control. b, Recombinant NLitself has no effect on cell migration. HUVECs migration assay wasperformed with indicated concentrations of ES, NL, and both of them,respectively, PBS is a control. c, HUVECs proliferation assay wasperformed with indicated concentrations of ES, NL, and Anti-NL, PBS as acontrol. The cell number was evaluated by MTT assay. d, Cell adhesionassay was performed with NL-deficient and control HMECs. NL-deficientcells were obtained by suppressing the expression of NL with RNAiplasmid BS/U6/1356. Control cells were transfected with blank plasmidBS/U6. Results are means±s.e.m., n=4 (a, d), n=5(b, c). e, Thesuppression of expression of NL by RNAi plasmids was demonstrated by IBusing Anti-NL. Plasmid BS/U6/1356 can suppress the expression of NL, butBS/U6/263 dose not work. The myosin blots serve as loading controls. f,Cell proliferation assay was performed with NL-deficient and controlHMECs. NL-deficient cells were obtained by suppressing the expression ofNL with RNAi plasmid BS/U6/1356. Control cells were transfected withblank plasmid pBS/U6. ES was added with indicated concentrations. Thenumber of cells was evaluated by MTT assay.

FIG. 4 shows that nucleolin mediates endostatin signal network. a-f, ESis internalized by HMECs. HMECs were incubated with or without (a) 10μg/ml biotinylated ES for 0.5 h (b), 1 h (c), 2 h (d), 3 h (e), and 7 h(f), respectively. The internalized ES was stained with TRITC-labeledavidin. g, The internalized ES was blocked by incubating cell withAnti-NL. Scale bar, 25 μm. h, In nuclei, ES inhibits CK2-mediated NLphosphorylation. Phosphorylation assay was performed as described inMethods. Phosphorylated NL was detected by SDS-PAGE and autoradiography.The NL blots serve as loading controls. i-k, Colocalization of cellsurface NL with integrin β1 on HMEC surface. Intact HMECs were stainedby mouse Anti-NL and rabbit Anti-integrin β1, and detected by indirectimmunofluorescence using laser scanning confocal microscopy. Scale bar,10 μm.

FIG. 5 shows the distribution of cell surface nucleolin depends on cellgrowth states. a-h, Distribution of cell surface NL on HMECs. Cellsurface NL of proliferating cells and quiescent cells was detected byindirect immunofluorescence using rabbit Anti-NL. DAPI indicates allcells in the field. Scale bar, 20 μm. Cell cycle positions ofproliferating cells (g) and quiescent cells (h) were detected by flowcytometry, respectively. Relatively quiescent cells were obtained byserum starvation for 24 h. i-l, Distribution of cell surface NL intumor-bearing nude mice. Immunohistochemistry was performed as describedin Methods. The blood vessels of heart (i), kidney (j), lung (k), andtumor (l) were indicated by arrows. Endothelial cells, which containcell surface NL, were stained brown. Scale bar, 50 μm.

FIG. 6 provides a working model for nucleolin-mediated endostatin signalnetwork. A big complex, which is composed of cell surface NL, integrin(such as integrin α5β1), and other proteins, is involved in the ESsignal network. NL binds to myosin, which links NL and actin filaments.Similarly, integrin binds indirectly to bundles of actin filaments viathe intracellular anchor proteins talin, α-actinin, filamin, andvinculin. No direct interaction can be observed between NL and integrin.ES binds to this complex competing with ECM, which leads to the decreaseof cell adhesion and migration. This binding can also triggerintegrin-mediated signal transduction. On the other hand, this complexcan mediate ES internalization, in which myosin serves as a transporter.Subsequently, ES may be released into cytoplasm. Some ES serve as aninhibitor of NL which can stabilize the mRNA of Bcl-2, and exert otherfunctions. The remaining ES is transported into nuclei, where itinhibits CK2-mediated NL phosphorylation as well as other down streamevents.

FIG. 7 shows the co-localization between ES and NL in vivo. BiotinylatedES and antibodies against NL were injected intravenously into micebearing B16-F10 tumors. Biotinylated ES and purified rabbit IgG wasinjected intravenously as a control. The distribution of thebiotin-labeled ES and anti-NL in heart (a-c), liver (d-f), kidney (g-i),tumor (j-l), and control tumor (m-o) were detected with bothTRITC-conjugated avidin and FITC-conjugated secondary antibodies. Scalebar, 50 μm.

FIG. 8 shows the kinetic binding sensorgrams depicting real-timeinteractions between ES and NL at indicated concentrations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention derives partly from the discovery that nucleolinacts as a specific receptor for ES and facilitates its function inantiangiogenesis.

NL is a ubiquitous, nonhistone protein, which was first isolated fromnucleolus. It is very interesting that the amount of NL is correlated tocell proliferation, which is regulated by Granzyme A and self-cleavingactivity. Nucleolin also undergoes self-cleavage, which is decreasedwhen cells enter a proliferative stage, as well as being cleaved byGranzyme A, an esterase secreted by cytotoxic lymphocytes (Chen et al.,J. Biol. Chem., 1991, 266, 7754-7758; Fang and Yeh, Exp. Cell. Res.,1993, 208, 48-53; Pasternack et al., J. Biol. Chem., 1991, 266,14703-14708). The cleavage and concomitant degradation of the proteinprovides for post-translational regulation of nucleolin.

As a multifunctional protein, NL exerts a critical and fundamentaleffect on cell proliferation, including organization of nucleolarchromatin, packaging of pre-RNA, rDNA transcription, and ribosomeassembly. These activities are regulated by certain protein kinases suchas casein kinase 2 (CK2) and cdc2 which are under strict control ofother cell cycle proteins. Moreover, NL also functions as a cell surfacereceptor, shuttling between cell surface, cytoplasm, and nucleus. As areceptor of many viruses and cytokines, NL triggers the internalizationof ligands as soon as these ligands bind to it.

Nucleolin has been described by Orrick et al (1973) as a protein withmolecular weight about 100-110 kDa, and mainly existing in the nucleusof the propagating cells. Nucleolin exhibits auto-degradation and showstwo degraded bands about 70 and 50 kDa in Western blotting analysis.Nucleolin is highly phosphorylated and methylated, and can beADP-ribosylated. Because synthesis of the nucleolin is positivelycorrelated with increased rate of cell division, tumor cells and rapidlydividing cells have higher levels of nucleolin content. The sequence ofNL was reported earlier in Srivastava, et al., Cloning and sequencing ofthe human nucleolin cDNA. FEBS Lett. 250 (1), 99-105 (1989)

Nucleolin (also known as P92 and C23) is the most abundant nucleolarphosphoprotein in actively growing cells (Srivastava et al., FEBS Lett.,1989, 250, 99-105; Srivastava et al., J. Biol. Chem., 1990, 265,14922-14931). It has been described by several groups and shown toparticipate primarily in ribosome biogenesis (Ghisolfi. et al., Mol.Biol. Rep., 1990, 14, 113-114; Sipos and Olson, Biochem. Biophys. Res.Commun., 1991, 177, 673-678) and transport of ribosomal components(Schmidt-Zachmann et al., Cell, 1993, 74, 493-504). Nucleolincontributes to ribosome biosynthesis by transiently binding to thepre-ribosomes in the nucleolus via a ribonucleoprotein consensussequence (Bugler et al., J. Biol. Chem., 1987, 262, 10922-10925;Ghisolfi-Nieto et al., J. Mol. Biol., 1996, 260, 34-53; Sapp et al.,Eur. J. Biochem., 1989, 179, 541-548). Here, nucleolin can represent upto 5% of the total nucleolar protein (Lapeyre et al., Proc. Natl. Acad.Sci. U.S.A., 1987, 84, 1472-1476; Sapp et al., Eur. J. Biochem., 1989,179, 541-548). However, it has also been shown to be involved incytokinesis, nucleogenesis, cell proliferation and growth,transcriptional repression, replication, signal transduction andchromatin decondensation reviewed in (Tuteja and Tuteja, Crit. Rev.Biochem. Mol. Biol., 1998, 33, 407-436).

The multifunctionality of this protein arises from the presence ofdistinct structural and functional domains within the protein (Creancieret al., Mol. Biol. Cell., 1993, 4, 1239-1250; Sapp et al., Eur. J.Biochem., 1989, 179, 541-548). Three domains have been described withinthe nucleolin protein, the N-terminal domain, the central domain and theC-terminal domain. Contained in the N-terminal domain are sequences thatshow homology with the high-mobility group (HMG) and are responsible forinteractions with chromatin (Erard et al., Eur. J. Biochem., 1988, 175,525-530). The central domain contains four RNA recognition motifs andbinds specifically with the short stem loop of the 18S and 28S ribosomalRNA (Bugler et al., J. Biol. Chem., 1987, 262, 10922-10925) while theC-terminal domain contains regions that are capable of unstacking basesin RNA (Ghisolfi et al., Mol. Biol. Rep., 1990, 14, 113-114;Ghisolfi-Nieto et al., J. Mol. Biol., 1996, 260, 34-53). Nucleolincontains a bipartite nuclear localization signal, spanning both theN-terminal and central regions of the protein, which facilitatestransport into the nucleus where nucleolin accumulates due tointeractions with other proteins (Schmidt-Zachmann and Nigg, J. CellSci., 1993, 105, 799-806).

The domain structure of nucleolin has led the protein to be classifiedas an Ag-NOR protein (Active ribosomal gene located in the NucleolarOrganizer Region) otherwise known as markers of active ribosomal genes(Roussel et al., Exp. Cell. Res., 1992, 203, 259-269). It has been shownthat transcription of ribosomal genes requires the presence of Ag-NORproteins and the expression of Ag-NOR proteins has been associated withthe prediction of tumor growth rate in cancers.

Nucleolin has also been purified as a matrix attachment region (MAR)binding protein from human erythroleukemia cells. In these studies,nucleolin was shown to participate in the anchoring of chromatin loopsto the nuclear matrix (Dickinson and Kohwi-Shigematsu, Mol. Cell. Biol.,1995, 15, 456-465).

Nucleolin is highly phosphorylated and has been shown to be a substratefor casein kinase II (Csermely et al., J. Biol. Chem., 1993, 268,9747-9752; Schneider and Issinger, Biochem. Biophys. Res. Commun., 1988,156, 1390-1397), Protein kinase C-.xi. (Zhou et al., J. Biol. Chem.,1997, 272, 31130-31137), and Cdc2 (Belenguer et al., Mol. Cell. Biol.,1990, 10, 3607-3618). Furthermore, the phosphorylation of nucleolin hasbeen shown to regulate the subcellular localization of the protein.

Nucleolin also undergoes self-cleavage, which is decreased when cellsenter a proliferative stage, as well as being cleaved by Granzyme A, anesterase secreted by cytotoxic lymphocytes (Chen et al., J. Biol. Chem.,1991, 266, 7754-7758; Fang and Yeh, Exp. Cell. Res., 1993, 208, 48-53;Pastemack et al., J. Biol. Chem., 1991, 266, 14703-14708). The cleavageand concomitant degradation of the protein provides forpost-translational regulation of nucleolin.

Anti-nucleolin antibodies have been found in the sera of patients withsystemic connective tissue diseases including systemic lupuserythromatosus (SLE) (Minota et al., J. Immunol., 1990, 144, 1263-1269;Minota et al., J. Immunol., 1991, 146, 2249-2252) and scleroderma-likechronic graft vs. host disease (Bell et al., Br. J. Dermatol., 1996,134, 848-854). The pharmacological modulation of nucleolin expressionmay therefore be an appropriate point of therapeutic intervention inpathological conditions.

Currently, there are no known therapeutic agents which effectivelyinhibit the synthesis of nucleolin. Consequently, there remains a longfelt need for agents capable of effectively inhibiting nucleolinfunction.

It is believed that expression level of nucleolin correlates with cellproliferation rate. Nucleolin levels are highest in tumors and moderatein other rapidly dividing cells. It can be used in studies of differentcancer cell lines as useful marker for cell proliferation. Sincenucleolin plays a vital role in tumor cell proliferation, the presentinvention provides a strategy of inhibition of nucleolin expression tosuppress the growth rate of tumor cells.

Endostatin (referred to herein as “ES”) is a 20 kDa C-terminal globulardomain of the collagen-like protein, collagen XVIII. It was originallyisolated from the supernatant of a cultured murine hemangioendotheliomacell line for its ability to inhibit the proliferation of capillaryendothelial cells. In animal tests, tumor dormancy was induced followingrepeated cycles of ES treatment without any drug resistance. Moreover,low toxicity of ES was observed in both animal tests and clinicaltrials. ES exhibits potent activities in inhibiting endothelial cellproliferation, migration, adhesion, survival, and in inducing cellapoptosis. Although integrins, tropomyosin, glypicans, and E-selectinare speculated as ES receptors associated with cell migration, andβ-catenin and Shb adaptor are involved in ES-induced endothelial cellsG1 arrest and apoptosis, the exact molecular mechanism of ES inantiangiogenesis is still in debate, and the reason for the low toxicityof ES in animal tests and clinical trials is still unknown. In addition,there is still a lack of adequate explanation for the fact that highconcentration of ES is required in order to achieve anti-tumor effect inboth animal tests and clinical trials.

The novel discovery, as disclosed here in the present invention, thatnucleolin is a specific receptor to which endostatin binds, and mediatesendostatin's function as an angiogenesis inhibitor provides a basis toscreen and generate additional small molecules which also can functionas angiogenesis inhibitor, and yet possesses additional properties thatare more readily adapted as compared to the protein endostatin.

It has been shown that in order for endostatin to function effectivelyas an angiogenesis inhibitor, a large quantity of endostatin must beproduced and administered into the cancer subjects, such as a mammal ora human, in order to produce the desired anticancer effect. Theproduction of endostatin can be prohibitively expensive due to thedemand of high dose in cancer therapy using endostatin. Thus, thereexists a great need to find alternative methods to find angiogenesisinhibitors other than endostatin, which can be used effectively andeconomically produced in reducing cancer growth.

In addition, in order to increase the sensitivity of tumor cells toendostatin, methods are disclosed in the present invention wherebyexogenous NL is introduced into target cells so that the cells willexpress higher than normal amount of surface NL. These modified targetcells are more sensitive to the action of ES molecules due to thepresence of high level of NL molecules, which as described above, act asthe specific receptor for ES.

In another aspect of the invention, antibodies against NL can be used toscreen those cancer cells or endothelial cells which express a highlevel of surface NL. Finding this particular group of patients isextremely beneficial to the effectiveness of angiogenesis-related cancertherapy, since patients with high expression of surface NL are ideal forthe administration of ES in order to inhibit their tumor growth.

As used herein, the term “endostatin” (also known as “ES”) refers to aprotein that is preferably 18 kDa to 21 kDa in size as determined bynon-reduced and reduced gel electrophoresis, respectively. The termendostatin also includes precursor forms of the 18 kDa to 20 kDaprotein. The term endostatin also includes fragments of the 18 kDa to 20kDa protein and modified proteins and peptides that have a substantiallysimilar amino acid sequence, and which are capable of inhibitingproliferation of endothelial cells. For example, silent substitutions ofamino acids, wherein the replacement of an amino acid with astructurally or chemically similar amino acid does not significantlyalter the structure, conformation or activity of the protein, is wellknown in the art. Such silent substitutions are intended to fall withinthe scope of the appended claims.

It will be appreciated that the term “endostatin” includes shortenedproteins or peptides wherein one or more amino acids is removed fromeither or both ends of endostatin, or from an internal region of theprotein, yet the resulting molecule retains endothelial proliferationinhibiting activity. The term “endostatin” also includes lengthenedproteins or peptides wherein one or more amino acid is added to eitheror both ends of endostatin, or to an internal location in the protein,yet the resulting molecule retains endothelial proliferation inhibitingactivity. Such molecules, for example with tyrosine added in the firstposition are useful for labeling such as radioiodination with ¹²⁵Iiodine for use in assays. Labeling with other radioisotopes may beuseful in providing a molecular tool for destroying the target cellcontaining endostatin receptors.

Similarly, as used herein, the term “nucleolin” (also known as “NL”)refers to a protein that is preferably 100 kDa (The exact MW of 80 kDawithout post-modification) in size as determined by reduced gelelectrophoresis. The term nucleolin also includes precursor forms of the100 kDa protein. The term nucleolin also includes fragments of 100 kDaprotein and modified proteins and peptides that have a substantiallysimilar amino acid sequence, and which are capable of inhibitingproliferation of endothelial cells. For example, silent substitutions ofamino acids, wherein the replacement of an amino acid with astructurally or chemically similar amino acid does not significantlyalter the structure, conformation or activity of the protein, is wellknown in the art. Such silent substitutions are intended to fall withinthe scope of the appended claims.

It will be appreciated that the term “nucleolin” includes shortenedproteins or peptides wherein one or more amino acids is removed fromeither or both ends of nucleolin, or from an internal region of theprotein, yet the resulting molecule retains its specific bindingactivity for ES. The term “nucleolin” also includes lengthened proteinsor peptides wherein one or more amino acid is added to either or bothends of nucleolin, or to an internal location in the protein, yet theresulting molecule retains the specific ES related NL activity. Suchmolecules, for example with tyrosine added in the first position areuseful for labeling such as radioiodination with ¹²⁵I iodine for use inassays.

The term “NL-specific” refers to the ability of NL binding to anangiogenesis inhibitor, and mediates its inhibition activity.

The term “angiogenesis-dependent” refers to those cancers and tumorswhose growth or migration require angiogenesis, including those thatwould require for their growth in either volume or mass, or both, anincrease in the number and density of the blood vessels supplying themwith blood.

As used herein, the term “subject” refers to any animal, such as amammal, including but not limited to a human, a non-human primate, arodent, a pig, a rabbit, and the like, which is to receive a particulartreatment, or undergoing a particular procedure such as screening forthe level of presence of a particular molecule.

As used herein, the term “sample” is used in its broadest sense,including, but not limited to a biological sample and environmentalsample. In one sense, it is meant to include a specimen or cultureobtained from any source, as well as biological and environmentalsamples. Biological samples may be obtained from animals (includinghumans) and encompass fluids, solids, tissues, and gases. Biologicalsamples include blood products, such as plasma, serum and the like.Environmental samples include environmental material such as surfacematter, soil, water, minerals, crystals and industrial samples. Suchexamples are not to be construed as limiting the sample typesencompassed by the present invention.

As used herein, the term “label” encompasses chemical or biologicalmolecules that are used in detecting the presence in a sample of atarget molecule which is capable of binding to or otherwise interactwith the label so as to indicate its presence in the sample, and theamount of the target molecule in the sample. Examples of such labelsinclude, but not limited to, a nucleic acid probe such as a DNA probe,or RNA probe, an antibody, a radioisotope, a fluorescent dye, and thelike.

As used herein, the term “usage instruction” includes instructions inthe kit for carrying out the procedure for detecting the presence of atarget molecular such as nucleolin in the sample to be tested. In thecontext of kit being used in the United States, the usage instructioncomprising the statement of intended use required by the U.S. Food andDrug Administration (FDA) in labeling in vitro diagnostic products. Itwould be apparent to one with ordinary skill in the art of medicaldiagnostic devices as to the format and content of these usageinstructions as required by the FDA.

As used in the present invention, an appropriate binding assay forselecting specific NL-related angiogenesis inhibitor includes HPLC,immunoprecipitation, fluorescent-binding assay, capillaryelectrophoresis, and so forth.

As used herein, an “anti-angiogenesis assay” is an experiment where apool of candidate molecules are screened in order to discover theeffectiveness of the candidate molecules in inhibiting angiogenesis. Inorder to discover whether a molecule has anti-angiogenesis property,various methods can be applied to carry out the present invention. Forexample, proteins and peptides derived from these and other sources,including manual or automated protein synthesis, may be quickly andeasily tested for endothelial proliferation inhibiting activity using abiological activity assay such as the bovine capillary endothelial cellproliferation assay. Other bioassays for inhibiting activity include thechick embryonic chorioallantoic membrane (CAM) assay, the mouse cornealassay, and the effect of administering isolated or synthesized proteinson implanted tumors. The chick CAM assay is described by O'Reilly, etal. in “Angiogenic Regulation of Metastatic Growth”, Cell, vol. 79 (2),Oct. 21, 1994, pp. 315-328, which is hereby incorporated by reference inits entirety. Additional anti-angiogenesis assays for screening forangiogenesis inhibitors can be found in Yu, et al., PNAS, Vol. 101, No.21, pp 8005-8010 (2004), which is hereby incorporated by reference inits entirety.

As used herein, the term “and/or” as used in the phrase “proliferationand/or migration” refers to two situations: 1) both proliferation andmigration of endothelial cells are modulated; 2) either proliferation ormigration, but not both, of endothelial cells are modulated.

As used herein, the term “link” refers to connecting antibody to acytotoxic agent such as a cytokine molecule, using conventional,well-known biological or chemical techniques such as cross-linking, andso forth.

The NL molecules can be used to generate polyclonal or monoclonalantibodies, which can in turn be used to both identify and quantify thelevel of NL in a particular target cells. NL labeled with appropriatemarkers such as radioisotopes and fluorescent dye can be used to for thedetection of endostatin in body fluids and tissues for the purpose ofdiagnosis or prognosis of angiogenesis-mediated diseases such as cancer.The present invention also includes methods of treating or preventingangiogenic diseases and processes including, but not limited to,arthritis and tumors by increasing the efficacy of endostatin in thoseangiogenesis-dependent cancers in a patient.

In some embodiments of the invention, methods such as flow cytometry aswell as Enzyme-linked Immunosorbent Assay (ELISA) techniques are usedfor quantification of the NL peptide.

Detection of the nucleic acid molecules of NL can be performed usingstandard molecular biological techniques such as DNA probehybridization, PCR, etc. General references for methods that can be usedto perform the various PCR and cloning procedures described herein canbe found in Molecular Cloning: A Laboratory Manual (Sambrook et al.,eds. Cold Spring Harbor Lab Publ. 1989, latest edition). Detection ofthe RNA molecules of NL can be performed using Northern blot analysis.Northern blot analysis involves the separation of RNA and hybridizationof a complementary labeled probe. In certain embodiments, RNA (orcorresponding cDNA) is detected by hybridization to an oligonucleotideprobe. A variety of hybridization assays using a variety of technologiesfor hybridization and detection are available. For example, in someembodiments, the TaqMan assay (PE Biosystems, Foster City, Calif.; Seee.g., U.S. Pat. Nos. 5,962,233 and 5,538,848, each of which is hereinincorporated by reference) is utilized. The assay is performed during aPCR reaction. The TaqMan assay exploits the 5′-3′ exonuclease activityof the AMPLITAQ GOLD DNA polymerase. A probe consisting of anoligonucleotide with a 5′-reporter dye (e.g., a fluorescent dye) and a3′-quencher dye is included in the PCR reaction. During PCR, if theprobe is bound to its target, the 5′-3′ nucleolytic activity of theAMPLITAQ GOLD polymerase cleaves the probe between the reporter and thequencher dye. The separation of the reporter dye from the quencher dyeresults in an increase of fluorescence. The signal accumulates with eachcycle of PCR and can be monitored with a fluorimeter.

In certain other embodiments, reverse-transcriptase PCR (RT-PCR) is usedto detect the expression of RNA. In RT-PCR, RNA is enzymaticallyconverted to complementary DNA or “cDNA” using a reverse transcriptaseenzyme. The cDNA is then used as a template for a PCR reaction. PCRproducts can be detected by any suitable method, including but notlimited to, gel electrophoresis and staining with a DNA specific stainor hybridization to a labeled probe. In some embodiments, thequantitative reverse transcriptase PCR with standardized mixtures ofcompetitive templates method described in U.S. Pat. Nos. 5,639,606,5,643,765, and 5,876,978 (each of which is herein incorporated byreference) is utilized.

Detection of the protein molecule of NL can be performed usingtechniques known in the art (e.g., radioimmunoassay, ELISA(enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitation reactions,immunodiffusion assays, in situ immunoassays (e.g., using colloidalgold, enzyme or radioisotope labels, for example), Western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.

For example, antibody binding is detected by detecting a label on theprimary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many methods are known in the art for detecting binding in animmunoassay and are within the scope of the present invention.

In certain cases, an automated detection assay is utilized. Methods forthe automation of immunoassays include those described in U.S. Pat. Nos.5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is hereinincorporated by reference. In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a prognosis based on the presenceor absence of a series of proteins corresponding to cancer markers isutilized.

Antibodies specific for NL and NL analogs are made according totechniques and protocols well known in the art. The antibodies may beeither polyclonal or monoclonal. The antibodies are utilized inwell-known immunoassay formats, such as competitive and non-competitiveimmunoassays, including ELISA, sandwich immunoassays andradioimmunoassays (RIAs), to determine the presence or absence of theendothelial proliferation inhibitors of the present invention in bodyfluids. Examples of body fluids include but are not limited to blood,serum, peritoneal fluid, pleural fluid, cerebrospinal fluid, uterinefluid, saliva, and mucus.

The present invention provides isolated antibodies that can be used inthe diagnostic kits in the detection of NL. In preferred embodiments,the present invention provides monoclonal antibodies that specificallybind to NL.

An antibody against NL in the present invention may be any monoclonal orpolyclonal antibody, as long as it can recognize the protein. Antibodiescan be produced by using NL or its analogue as the antigen usingconventional antibody or antiserum preparation processes.

The present invention contemplates the use of both monoclonal andpolyclonal antibodies. Any suitable method may be used to generate theantibodies used in the methods and compositions of the presentinvention, including but not limited to, those disclosed herein. Forexample, for preparation of a monoclonal antibody, protein, as such, ortogether with a suitable carrier or diluent is administered to an animal(e.g., a mammal) under conditions that permit the production ofantibodies. For enhancing the antibody production capability, completeor incomplete Freund's adjuvant may be administered. Normally, theprotein is administered once every 2 weeks to 6 weeks, in total, about 2times to about 10 times. Animals suitable for use in such methodsinclude, but are not limited to, primates, rabbits, dogs, guinea pigs,mice, rats, sheep, goats, etc.

For preparing monoclonal antibody-producing cells, an individual animalwhose antibody titer has been confirmed (e.g., a mouse) is selected, and2 days to 5 days after the final immunization, its spleen or lymph nodeis harvested and antibody-producing cells contained therein are fusedwith myeloma cells to prepare the desired monoclonal antibody producerhybridoma. Measurement of the antibody titer in antiserum can be carriedout, for example, by reacting the labeled protein, as describedhereinafter with the antiserum and then measuring the activity of thelabeling agent bound to the antibody. The cell fusion can be carried outaccording to known methods, for example, the method described by Koehlerand Milstein (Nature 256:495 [1975]). As a fusion promoter, for example,Sendai virus (HVJ) or, preferably, polyethylene glycol (PEG), is used.

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen (an antigen against theprotein) and a carrier protein is prepared and an animal is immunized bythe complex according to the same manner as that described with respectto the above monoclonal antibody preparation. A material containing theantibody against is recovered from the immunized animal and the antibodyis separated and purified.

The present invention provides for a method of inhibiting theproliferation and/or migration of endothelial cells by producing acombination antibody containing an anti-NL antibody linked to acytotoxic agent such as a chemokine, i.e. a Tumor Necrosis Factor-alpha,etc. When such a combination antibody is administered to a cell sampleincluding an endothelial cells, the anti-NL antibody will direct thetoxic agent to the endothelial cells, and thus bring the toxic agentsuch as Tumor Necrosis Factor-alpha to act upon the endothelial cells,and killing the cell growth.

Methods of linking an antibody to a second agent such as a cytotoxicagent in order to form a combination antibody, also know as animmunotoxic, is well known in the art. Two major advances in theimmunotoxin field have been the use of the recombinant DNA technique toproduce recombinant toxins with better clinical properties and theproduction of single-chain immunotoxins by fusing the DNA elementsencoding combining regions of antibodies, growth factors, or cytokinesto a toxin gene.

First-generation immunotoxins were constructed by coupling toxins to MAbor antibody fragments using a heterobifunctional cross-linking agent. Itwas also discovered that genetic engineering could be used to replacethe cell-binding domains of bacterial toxins with the Fv portions ofantibodies or with growth factors.

As well known in the art, cytokines are small protein molecules that arethe core of communication between immune system cells, and even betweenthese cells and cells belonging to other tissue types. They are activelysecreted by immune cells as well as other cell types. Cytokines that areproduced by immune cells form a subset known as lymphokines. Theiraction is often local, but sometimes can have effects on the whole body.

There are a lot of known cytokines that have both stimulating andsuppressing action on lymphocyte cells and immune response. Some of thebetter known cytokines include: histamine, prostaglandin, TNF-α, IL-1,and IL-6. There are three classes of cytokines.

The present invention provides kits for the detection andcharacterization of nucleolin in cancer diagnostics. In someembodiments, the kits contain antibodies specific for NL, in addition todetection reagents and buffers. In other embodiments, the kits containreagents specific for the detection of mRNA or cDNA (e.g.,oligonucleotide probes or primers) of NL. In preferred embodiments, thekits contain all of the components necessary to perform a detectionassay, including all controls, directions for performing assays, and anynecessary software for analysis and presentation of results.

Kits containing labels such as antibodies again NL for measurement of NLare also contemplated as part of the present invention. Antibodysolution is prepared such that it can detect the presence of NL peptidesin extracts of plasma, urine, tissues, and in cell culture media arefurther examined to establish easy to use kits for rapid, reliable,sensitive, and specific measurement and localization of endostatin.These assay kits include but are not limited to the followingtechniques; competitive and non-competitive assays, radioimmunoassay,bioluminescence and chemiluminescence assays, fluorometric assays,sandwich assays, immunoradiometric assays, dot blots, enzyme linkedassays including ELISA, microtiter plates, antibody coated strips ordipsticks for rapid monitoring of urine or blood, andimmunocytochemistry. For each kit the range, sensitivity, precision,reliability, specificity and reproducibility of the assay areestablished according to industry practices that are commonly known toand used by one with ordinary skill in the art.

Similarly, a diagnostic kit in the present invention can be used forlocalization of endostatin in tissues and cells. This NLimmunohistochemistry kit provides instructions, NL molecules, preferablylabeled and linked to a fluorescent molecule such as fluoresceinisothiocyanate, or to some other reagent used to visualize the primaryantiserum. Immunohistochemistry techniques are well known to thoseskilled in the art. This NL immunohistochemistry kit permitslocalization of endostatin in tissue sections and cultured cells usingboth light and electron microscopy. It is used for both research andclinical purposes. For example, tumors are biopsied or collected andtissue sections cut with a microtome to examine sites of endostatinproduction. Such information is useful for diagnostic and possiblytherapeutic purposes in the detection and treatment of cancer.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLES Example One

Nucleolin is an Endostatin-Binding Protein

To investigate the mechanism of action of ES, we isolated ES-bindingproteins directly from the membrane proteins of human microvascularendothelial cells (HMECs) with immobilized ES. Nucleolin was identifiedas a critical member in the ES signaling network, and is the mostinteresting one in the network. Here we describe that NL serves as anovel receptor for ES, and mediates the activity of ES inantiangiogenesis.

Methodology:

Methods that are used in the study of NL and ES interactions aregenerally known in the art. Descriptions of these methods are providedas follows:

Cell Migration Assay

Endothelial cells (HMECs or HUVECs, 2×10⁴ per well) were seeded into theupper chamber of Transwell™ filter (8 μm pores, Costar) with DMEM mediumcontaining 0.5% FCS and 10 ng/ml VEGF (PeproTech EC). ES (from Protgen)with indicated concentrations and other reagents (NL and Anti-NL) wereadded in both upper chamber and lower chamber at the beginning of themigration assay. The endothelia cells were allowed to migrate for 6 h at37° C. and 5% CO₂. After fixed with ethanol and stained with Eosin, thecells migrated completely through the filter to the lower chamber werecalculated as the average number of cells observed in five randomhigh-power (×400) fields per well in triplicate wells.

Cell Proliferation Assay

Endothelia cells (HMECs or HUVECs, 1×10³ per well) were seeded in a96-well plate with DMEM medium containing 0.5% FCS and 10 ng/ml bFGF. ESwith indicated concentrations and other reagents were added at thebeginning of the proliferation assay with a final volume of 200 μl. Theendothelial cells were allowed to proliferate for 48 h at 37° C. and 5%CO₂. After 48 h incubation, the medium was replaced with DMEM mediumwithout phenol red and 0.5 mg/ml MTT with a final volume of 100 μl. Thecells were incubated further for 4 h at 37° C. and 5% CO₂. Subsequently,the cells were lysed with iso-propanol containing 0.05 M hydrochloride,and the absorbance at 570 nm was measured.

Identification of Isolated Proteins with MALDI-TOF Mass Spectrometry

Fractions obtained from the ES-Ni-NTA affinity column were applied to12% SDS-PAGE, and the major bands were digested using sequencing gradeporcine-modified trypsin (Promega). The peptides produced were analyzedby MALDI-TOF mass spectrometry using a Bruker Biflex lineartime-of-flight spectrometer (Bruker Franzen), equipped with a multiprobeSCOUT source, ultra-nitrogen laser (337 nm), and a dualmicrochannel-plate detector. The MALDI-TOF data was searched againstSwiss-Prot protein database for protein identification.

Indirect Immunofluorescence

The HMECs were incubated with ES (20 μg/ml) for 1 h at 37° C. and 5%CO₂. Without permeabilized, the HMECs were stained with indicatedantibodies. FITC-linked goat anti-mouse IgG and TRITC-linked goatanti-rabbit IgG were used as the secondary antibodies. Confocalfluorescence imaging was performed on an Olympus Fluoview laser scanningconfocal imaging system (Olympus Inc.). Images were captured usingmultiple photomultiplier tubes regulated by Fluoview 2.0 software(Olympus).

Production of Recombinant Nucleolin

The cDNA of NL was obtained from HMECs by RNA isolation and reversetranscription system (Promega). The sequence of NL fusing with apolyhistidine (His)₆ tag was subcloned into pPIC9K (Invitrogen). Thisplasmid was linearized with restriction enzyme Sal I (Promega) andelectrotransformed into pichia strain GS115. A stable transformant wasselected using G418 (Invitrogen) and then grown in a 30° C. shakingflask for 3 days in BMMY medium as described by the manufacturer. Thesupernatant was obtained and NL was purified with Ni-NTA nickelion-affinity columns (Qiagen).

Preparation of Polyclonal Antibodies Against Nucleolin

New Zealand White rabbits were immunized with 50 μg of recombinant NLprepared by pPIC9K-GS115 pichia expression system as describedpreviously. The initial subcutaneous injection was administered inFreund's complete adjuvant, and a booster dose of 50 μg was givenintramuscularly on day 14 in Freund's incomplete adjuvant. Boosts of 50μg of NL were given subcutaneously without any adjuvant at 4, 10, 16,and 22 weeks. At 1 week after the last boost, serum was obtained, andthe IgG was purified by affinity chromatography on a protein A column(Amersham Biosciences), eluted with glycine-HCl buffer (0.15 M, pH 2.5),and immediately neutralized with 0.15 M Tris to a pH of 6.8-7.2. Thecombined fractions were filter-sterilized (0.2 μM), and aliquots of theantibodies preparation were stored at −80° C.

Cell Adhesion Assay

Cells to be tested were serum-starved for 30 min, and then seeded in a96-well plate which is coated with endostatin (20 μg/ml) and polylysine(50 μg/ml). The plate was incubated for 1 h at 37° C. and 5% CO₂.Unbounded cells were removed by washing with fresh medium gently. Theremained cells were stained with crystal violet (0.1% in ddH₂O) for 25min at RT. The plated was washed with tape water, and the remainedcrystal violet was solubilized with 0.5% Triton X-100 (diluted inddH₂O). The absorbance at 570 nm was measured.

As the targets of many angiogenesis inhibitors, endothelial cells areused as models for antiangiogenesis assays in vitro. Unfortunately, theresults are usually variable and inconsistent, probably due to the factthat the assays are not well established and the cell lines are not sostable. To this end, HMEC cell line was chosen that meets the criteriaas a good model for antiangiogenesis assay. In the cell migration assay,ES inhibited the migration of HMECs stimulated by VEGF in a dosedependent manner and the half-maximal inhibitory concentration (IC50)was observed at 4 μg/ml (FIG. 1 a). ES inhibited the migration of HMECsby 15% even at a concentration as low as 4 ng/ml (data not shown).Similar results were also obtained in the proliferation assay of HMECswith bFGF as a stimulus (FIG. 1 b). The results from both the cellmigration assay and the proliferation assay demonstrate that HMECs arevery sensitive to ES such that reliable and consistent results can beobtained.

The sensitive response of HMECs to ES implies that HMECs may containpotential receptor(s) for ES. Moreover, ES can specifically bind HMECsunder physiological conditions (see FIG. 2 d below). We thus started toisolate ES receptor(s) from HMECs. Recombinant ES was pre-loaded on toNi-NTA beads via its N-terminal His-tag. Crude fraction of plasmamembrane of HMECs was prepared and treated with 1% Triton X-100 torelease proteins from plasma membrane as described by Marshak et al. Theprepared plasma membrane proteins were applied to the ES-Ni-NTA beads.Unbound proteins were removed from ES-Ni-NTA beads with PBS buffer.Every fraction was collected and subjected to analysis by reducingSDS-PAGE. The control was carried out in parallel, and the onlydifference was substituting Ni-NTA for ES-Ni-NTA. Two proteins withapparent molecular weights of 110 kDa and 80 kDa showed specific bindingability to ES-Ni-NTA beads (FIG. 2 a), and were subsequently identifiedas NL (110 kDa) and its degraded fragment (80 kDa) by peptide massfingerprinting using matrix-assisted laser desorption/ionization-time offlight-mass spectrometry (MALDI-TOF). The identity of NL was furtherverified by immunoblotting (IB) with monoclonal antibody against NL(FIG. 2 a). To verify the interaction between ES and NL, the followingstudies were carried out. The results of immunoprecipitation (IP) invitro show that the interaction between ES and recombinant NL isspecific (FIG. 2 b), and a complex is formed between ES and NL. Thiscomplex can be disrupted by 200 mM heparin (FIG. 2 c). ES can also bindHMECs via NL because polyclonal antibodies against NL can block suchbinding (FIG. 2 d). This conclusion was further verified by IP, whichwas carried out with ES-preincubated HMECs (FIG. 2 e). The results showthat ES and total endogenous NL (cell surface, cytoplasma, and nucleus)form a complex in living cells. Moreover, co-localization between ES andNL on cell surface of HMECs was also observed by laser scanning confocalimmunofluorescence microscopy (FIG. 2 f-i). Taken together, ESspecifically binds to NL both in vitro and in vivo, which indicates thatNL is a potential receptor for ES.

Example Two

Nucleolin is a Novel Receptor for Endostatin

If NL is a receptor for ES, it should mediate the activities of ES inantiangiogenesis such as inhibiting the migration, proliferation, andadhesion of endothelial cells. To characterize the role of NL during theprocess of mediating ES activities, competitive cell migration andproliferation assays were therefore performed with ES, recombinant NL,and polyclonal antibodies against NL, respectively. Since NL wasisolated from HMECs as a potential receptor for ES in antiangiogenesis,whether or not NL plays similar roles in other widely acceptedendothelial cells should be demonstrated. To this end, human umbilicalvein endothelial cell (HUVEC), isolated directly from umbilical veins,were subjected to competitive cell migration and proliferation assays.This kind of endothelial cells can migrate through a microporous (8 μm)membrane under stimulation of VEGF, and ES inhibits such migration.Recombinant NL lifted the inhibition of ES in the cell migration assayin a dose dependent manner (FIG. 3 a), indicating that recombinant NL isinvolved in the antiangiogenesis activities of ES. The possibility thatNL itself has a stimulating activity on the migration of endothelialcells was excluded because recombinant NL itself had no effect on cellmigration as shown in FIG. 3 b. Similar results were also obtained whenthe cell proliferation assay was performed with HUVECs (FIG. 3 c). Notsurprisingly, polyclonal antibodies against NL blocked the inhibition ofES on cell proliferation (FIG. 3 c). In addition, as shown in FIG. 3 f,where cell proliferation assay was performed with NL-deficient cells andcontrol HMECs, it can be seen that the anti-angiogenesis effect of ESrequired the presence of NL molecule in the target cells. All thesestudies strongly suggest that NL is a receptor for ES inantiangiogenesis.

To further confirm that NL is the receptor for ES, the expression of NLwas suppressed by RNA interference (RNAi), followed by evaluating such achange on cell adhesion, another important activity of ES inantiangiogenesis. The results show that adhesions of HMECs on bothimmobilized ES and immobilized polylysine, a synthetic extracellularmatrix, significantly decreased when the expression of NL was suppressedby DNA vector-based RNAi (FIG. 3 d), even though the expression of NLwas not suppressed completely (FIG. 3 e). Rehn et al. reported that theadhesion of endothelial cells on immobilized ES is critical for theactivities of ES such as formation of focal adhesions andphosphorylation of FAK, and loss of this adhesion implies loss offunctions of ES on endothelial cells. Along the lines of these studies,our results demonstrate that NL is essential for the activities of ESand the adhesion of endothelial cells to extracellular matrix (ECM). Insum, we conclude that NL is a novel receptor for ES and plays a key rolein the signal transduction pathways of ES.

Example Three

Nucleolin Mediates Endostatin Signal Pathway

To unravel the exact role of NL in the signal transduction pathways ofES, we then investigated the down stream events. Retrospect that theamount of ES-NL complex varied when incubating ES with HMECs, andreached the maximal level around 2 h (FIG. 2 e). ES may be internalizedby HMECs via cell surface NL, and some of the internalized ES isdegraded by HMECs subsequently. It seems that there is a balance betweenES internalization and degradation (FIG. 2 e).

To confirm that the internalization of ES is via cell surface NL,immunofluorescence localization was carried out with biotinylated ES.HMECs were incubated with biotinylated ES for different periods of time,and the process of internalization of ES was observed under fluorescencemicroscope after biotinylated ES was stained with TRITC-labeledstreptavidin (FIG. 4 a-f). Upon 30 min of incubation, most of theinternalized ES distributed in cytoplasm but the amount was small (FIG.4 b). Upon 1 h of incubation, the internalized ES increased and began toaccumulate in nuclei (FIG. 4 c). The amount of internalized ES reachedthe maximum at about 2 h of incubation (FIG. 4 d). By 3 h, thenucleus-accumulated ES began to disappear (FIG. 4 e). By 7 h, little EScould be found in nuclei (FIG. 4 f). There was a lag phase in thefluorescence assay (FIG. 4 b-d) comparing with IP (FIG. 2 e) for theamount of internalized ES to reach their maximals, probably thedegradation rate of ES is faster than that of biotinylated ES.Importantly, the amount of internalized biotinylated ES wassignificantly reduced when HMECs were preincubated with polyclonalantibodies against NL, demonstrating that ES is internalized via cellsurface NL (FIG. 4 d, g). Moreover, some small light dots inside thecells were observed under fluorescence microscope (FIG. 4 b-e), whichmay suggest that ES is internalized in vesicles in the process ofinternalization. This observation is consistent with previous reports byWickstrom and Christian et al. To explain how NL mediates theinternalization of ES, cross-linking experiments and IP were carried,respectively, during the process of internalization of ES. At first, weexpected to isolate the complex of ES, NL, and other proteins in livingHMECs with cross-linking reagents. A huge complex with a molecularweight far more than 300 kDa was thus obtained using BS3 (across-linking reagent from PIERCE Ltd.) (data not shown). Although thiscomplex can be stained by antibodies against either ES or NL, none ofthe components could be identified by this method. Alternatively, IPwith polyclonal antibodies against NL was introduced into lysed HMECs toisolate the proteins which interact directly with NL. A protein with amolecular weight around 200 kDa was thus found, and it was identified asnon-muscle myosin by PMF using MALDI-TOF. Myosins constitute a hugesuperfamily, which is involved in membrane dynamics and actinorganization at cell cortex, thus affecting cell migration, adhesion,and endocytosis. Within this superfamily, two-head class-V myosins canserve as transporters for vesicles, organelles, and mRNA particles alongactin filaments. We also found that cell surface NL can bind ES andtransport ES into cell nuclei (FIG. 4 a-f). Since myosin is anintracellular protein, it must bind to the intracellular domain of cellsurface NL. We speculate that this NL-myosin complex may serve as atransporter during the process of ES internalization. Similar processwas also reported by Shibata et al. that midkine, a growth factor inneural cells, can also be internalized and located into nuclei via NL.Interestingly, antibodies against the N-terminus of NL can beinternalized by Hep-2 cells via cell surface NL. Therefore,internalization appears to be a general phenomenon and is inevitablewhen a ligand binds to cell surface NL. The difference is that ES caninhibit cell proliferation whereas antibodies against NL cannot (FIG. 3c).

These observations indicate that although many ligands can bind to cellsurface NL specifically and trigger their internalizations, their fatesafter internalization are different.

The functions of ES were then investigated when it was transported tonuclei. Bouche et al. reported that NL can promote rDNA transcriptionand ribosome biogenesis in nuclei, which are critical for cell survivaland proliferation, as long as its serine residues in the N-terminus isphosphorylated by some kinds of kinases such as casein kinase 2 (CK2)under the stimulation of bFGF. Folkman and his colleagues also reportedthat proliferation of endothelial cells, which is stimulated by bFGF,could be inhibited by ES. According to the studies of Bouche et al., thebFGF-stimulated phosphorylation of NL is mediated by CK2 but not by anyother kinases such as cdc2 in this isolated-nuclei system. Therefore, wespeculate that ES inhibits cell proliferation by inhibiting theCK2-mediated phosphorylation of NL, which is stimulated by bFGF. Toconfirm this hypothesis, nuclei from quiescent HMECs were isolated andthe phosphorylation assay was carried out with or without ES. Theresults show that phosphorylation of NL was inhibited when the nuclei ofHMECs were preincubated with ES (FIG. 4 h). Moreover, phosphorylation ofNL is stimulated by bFGF rather than VEGF (FIG. 4 h), which may providean explanation for previous reports that bFGF can stimulateproliferation of endothelial cells, whereas VEGF can stimulate migrationof endothelial cells. Our observations demonstrate that ES inhibitsbFGF-stimulated phosphorylation of NL which is mediated by CK2,consequently, inhibits cell survival and proliferation.

Example Four

ES Affects Cell Motility and Adhesion via NL-Myosin Complex

We have demonstrated so far that NL is a novel receptor for ES ininhibiting cell migration, proliferation, and adhesion. Since integrinwas also reported to be a receptor for ES, it is interesting to testwhether there is an interaction between NL and integrin. Among theintegrin family, integrin α5β1 was reported to be the receptor for ES byRehn et al. and Sudhakar et al., therefore, colocalization between cellsurface NL and integrin α5β1 was performed by indirectimmunofluorescence using the mouse anti-integrin β1 and the rabbitanti-NL. Certain overlap between cell surface NL and integrin β1 wasobserved by laser scanning confocal microscopy (FIG. 4 i-k), suggestingthere are some interactions between NL and integrin β1 on cell surface.Consequently, IP with polyclonal antibodies against NL was introduced tocapture the complex of NL and integrin 1. Unfortunately, integrin β1cannot be detected in the precipitate, implying that the interactionbetween NL and integrin β1 is indirect. Previous studies demonstratedthat myosin is involved in membrane dynamics and actin organization atcell cortex, thus affecting cell migration, adhesion, and endocytosis.We also demonstrates that NL and non-muscle myosin form a complex atcell cortex, and this complex is critical for the motility and adhesionof endothelial cells. Therefore, we speculate that ES may interfere withcell motility and adhesion mediated by NL-myosin complex. It isplausible that cell surface NL and integrin α5β1 along with otherproteins such as myosin form a huge complex which together serves as anintegrated receptor for ES (see FIG. 6).

Example Five

The Distribution of NL Provides a Basis for the Low Toxicity ofEndostatin

Endostatin specifically inhibits angiogenesis and tumor growth, and notoxicity on animal tests and only low toxicity on clinical trials of ESwere observed. The exact molecular mechanism behind these observationsis still unknown. Our conclusion that NL mediates the specificactivities of ES in antiangiogenesis seems to contradict the previousreport that NL is a ubiquitous protein in cells. To illuminate thisparadox, the abundance of cell surface NL on HMECs at different growthstates was investigated. Not surprisingly, the results show that theabundance of cell surface NL is much higher in proliferating cells thanthat of relatively quiescent cells (FIG. 5 a-f). The relativelyquiescent cells were obtained by serum starvation for 24 h, and theposition of cell cycle was detected by flow cytometry (FIG. 5 g-h). Theresults of flow cytometry show that the proportion of the G1 phaseincreases by 24%, and the S phase decreases by 30% after serumstarvation (FIG. 5 g-h). Although the cells are not completelyG1-arrested, the amount of cell surface NL decreased significantly afterserum starvation for 24 h. We speculate that this different abundance ofcell surface NL may result in the different sensitivity of endothelialcells in response to ES.

In order to investigate whether the similar cellular phenomena can alsobe found on animal studies, the abundance of cell surface NL in tumortissues and normal organs were measured. Polyclonal antibodies againstNL were injected into the subcutaneous dorsal of tumor-bearing nude miceat a site remote from the inoculated tumor, and the immunohistochemistrywas performed to determine the localizations of these antibodies (FIG. 5i-l). The results show that antibodies against NL are accumulated onlyin tumor-induced endothelial cells but not in normal tissues such asheart, kidney, and lung (FIG. 5 i-l). These discoveries are perfectlyconsistent with the antiangiogenesis theory of Folkman: in the adult,the endothelial cells of blood vessel are in a quiescent state with alittle turnover except that they are up-regulated by some endogenousangiogenic stimuli, and the proliferating endothelial cells appear insome physiological or pathological angiogenic processes such as tumorgrowth and metastasis. In sum, the different abundance of cell surfaceNL, the receptor for ES, on endothelial cells provides an explanationfor the low toxicity of ES in animal tests and clinical trials: sincethe surface NL of endothelial cells in tumor-induced blood vessels ismuch more abundant than that of normal organs, ES thus specificallybinds to NL and exerts its antiangiogenesis function selectively ontumor tissues; on the other hand, ES seldom bind to normal organsbecause they do not have much NL on their cell surface, which in turndoes not induce toxicity upon ES treatment.

Example Six

Recombinant nucleolin modulates the function of endostatin in cellmigration assay

The cDNA of NL was obtained from human microvascular endothelial cells(HMECs) using the SV total RNA isolation system and reversetranscription system (Promega) following the manufacture's protocol. Thesequence of NL fusing with polyhistidine (His)₆ tag was amplified by PCRand then subcloned into pPIC9K (Invitrogen). Following the manufacture'sprotocol, this plasmid was linearized with restriction enzyme Sal I(Promega) and electrotransformed into yeast strain GS115. A stabletransformant was selected using G418 and then grown in a 30° C. shakingflask for 3 days in BMMY medium (10 g/L yeast extract; 20 g/L peptone;100 mmol/L potassium phosphate, pH 6.0; 13.4 g/L yeast nitrogen base; 40mg/L biotin; and daily additions of methanol to the medium to yield afinal concentration of 0.5%). The supernatant was obtained and the NLwas purified on Ni-NTA nickel ion-affinity columns (Qiagen). We adjusted1 L of supernatant to pH 8.0 and applied it to a column with 6 ml Ni-NTAbeads. The column was washed and eluted according to the manufacturer'sinstructions. About 3 mg NL per liter was obtained by this procedure.Polyclonal antibodies against nucleolin were prepared with this protein.

Human umbilical vein endothelial cell (HUVEC) (2×10⁴ per well) wereseeded into the upper chamber of Transwell filter (8 μm pores, Costar)with DMEM medium, 0.5% FCS, and 10 ng/ml bFGF (PeproTech EC). ES (5μg/ml, Protgen) and rh-nucleolin (20 μg/ml), or antibodies againstnucleolin (20 μg/ml) were added at the beginning of the migration assay.The PBS was added into this migration assay system as a control. Thesame DMEM medium and reagents were added to the lower chamber. Theendothelia cells were allowed to migrate for 6 h at 37° C. and 5% CO₂.After fixed and stained with ethanol and Eosin, the cells migratedcompletely through the filter to the lower chamber were counted in fivedifferent areas under the optical microscope, and the averaged numberwas obtained. The results show that recombinant human nucleolinalleviated the inhibition of ES in the cell migration assay in a dosedependent manner, indicating that recombinant NL is involved in theantiangiogenesis activities of ES. Similarly, polyclonal antibodiesagainst NL blocked the inhibition of ES on cell proliferation.

Example Seven

Acceleration of Tumor Growth when Cell Surface Nucleolin was Blockedwith Antibodies Against Nucleolin.

The cDNA of NL was obtained from human microvascular endothelial cells(HMECs) using the SV total RNA isolation system and reversetranscription system (Promega) following the manufacture's protocol. Thesequence of NL fusing with polyhistidine (His)₆ tag was amplified by PCRand then subcloned into pPIC9K (Invitrogen). Following the manufacture'sprotocol, this plasmid was linearized with restriction enzyme Sal I(Promega) and electrotransformed into yeast strain GS115. A stabletransformant was selected using G418 and then grown in a 30° C. shakingflask for 3 days in BMMY medium (10 g/L yeast extract; 20 g/L peptone;100 mmol/L potassium phosphate, pH 6.0; 13.4 g/L yeast nitrogen base; 40mg/L biotin; and daily additions of methanol to the medium to yield afinal concentration of 0.5%). The supernatant was obtained and the NLwas purified on Ni-NTA nickel ion-affinity columns (Qiagen). We adjusted1 L of supernatant to pH 8.0 and applied it to a column with 6 ml Ni-NTAbeads. The column was washed and eluted according to the manufacturer'sinstructions. About 3 mg NL per liter was obtained by this procedure.Polyclonal antibodies against nucleolin were prepared with this protein.

Hela cells were inoculated in the subcutaneous space of nude mice.Starting from the next day, antibodies against NL were injected slowlyinto the subcutaneous dorsal of mice at a site remote from theinoculated tumors every 3 days. After the seventh injection, the nudemice were killed, and the tumors were weighed and three measurements ofthe tumor were taken. The results of these animal tests show that thetumor growth was accelerated significantly when the cell surfacenucleolin was blocked with antibodies against nucleolin. These resultsalso demonstrated that nucleolin play a key role in regulating the tumorgrowth and angiogenesis.

Example Eight

Screening for NL-Specific Angiogenesis Inhibitors Using ES-NI-NTAAffinity Chromatography

The cDNA of NL is obtained from human microvascular endothelial cells(HMECs) using the SV total RNA isolation system and reversetranscription system (Promega) following the manufacture's protocol. Thesequence of NL fusing with polyhistidine (His)₆ tag is amplified by PCRand then subcloned into pPIC9K (Invitrogen). Following the manufacture'sprotocol, this plasmid is linearized with restriction enzyme Sal I(Promega) and electrotransformed into yeast strain GS115. A stabletransformant is selected using G418 and then grown in a 30° C. shakingflask for 3 days in BMMY medium (10 g/L yeast extract; 20 g/L peptone;100 mmol/L potassium phosphate, pH 6.0; 13.4 g/L yeast nitrogen base; 40mg/L biotin; and daily additions of methanol to the medium to yield afinal concentration of 0.5%). The supernatant is obtained and the NL waspurified on Ni-NTA nickel ion-affinity columns (Qiagen). We adjust 1 Lof supernatant to pH 8.0 and applied it to a column with 6 ml Ni-NTAbeads. The column is washed and eluted according to the manufacturer'sinstructions. About 3 mg NL per liter is obtained by this procedure.This nucleolin is fixed on the Ni-NTA nickel ion-affinity columns(Qiagen) via its fusion peptide at the N-terminus. This affinity beadcan be used to screen the nucleolin-binding proteins in ahigh-throughput manner. The nucleolin-binding proteins can be identifiedby PFM with MALDI-TOF. The bioactivities of these nucleolin-bindingproteins in antiangiogenesis are detected with cellular experiment suchas cell migration assay and cell proliferation assay as described above.

Example Nine

Col-Localization of ES and NL on the Surface of Tumor Blood Vessels

Method: Surface Plasmon Resonance

Binding kinetics was determined by SPR using a BIAcore 2000™ (AmershamPharmacia Biotech) biosensor system. Purified ES was diluted to 100μg/ml in 20 mM sodium acetate, pH 6.5, and covalently immobilized on theresearch CM5 sensor chips using the amine coupling kit(1-ethyl-3-(dimethylaminopropyl)carbodiimide, (N-hydroxysuccinimide)according to the manufacturer. ES (100 μg/ml) in sodium acetate (pH 6.5)was injected until a response difference of 9,000 units was obtained.The unreacted moieties on the surface were blocked with ethanolamine (pH8.5; BIAcore AB). The SPR analysis between ES and NL was performed at25° C. with a 20 μl volume of NL (62.5, 125, 250, and 500 nM) in a flowbuffer HBS (10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactantP20, pH 7.4; BIAcore AB) at a flow rate of 10 μl/min. Time course of NL,free in flow buffer, were continuously recorded as resonance units (RU).The surface was regenerated by several 10 μl pulses of 100 mM NaOH (or100 mM HCl) flowing at 10 μl/min. The primary data were analyzed usingthe BIAevaluation 3.1 Software (BIAcore AB), applying a Langmuir bindingmodel (stoichiometry of 1:1) to calculate k_(a) (association rateconstant M⁻¹s⁻¹), k_(b) (dissociation rate constant, M⁻¹ s⁻¹), and K_(D)(equilibrium constant) for the interaction of ES with NL.

The binding affinity between ES and NL was determined by real-timesurface plasmon resonance (SPR), which is a rapid and sensitive methodfor evaluating the affinities involved in bimolecular interaction. Theequilibrium constant (K_(D)) for the interaction of ES with NL isderived to be 2.32×10⁻⁸ M from these curves.

Co-Localization In Vivo

Exponential growing B16/F10 mouse melanoma cells (2×10⁶ cells in 200 μlof PBS) were inoculated in the subcutaneous space of 2-mo-old Balb/cmice. The animals were used for experiments of co-localization betweenES and NL in vivo 8 days after the implantation. The biotinylated ES (40μg) and polyclonal rabbit antibodies against NL (200 μg) were injectedi.v., respectively. The biotinylated ES (40 μg) and purified rabbit IgGwas intravenously injected as a control. The mice were anesthetized 1hour after the injection, perfused through the heart with 20 ml PBS, andkilled. Some tissues and tumor of the mice were fixed and sectioned. Thesections were detected with both TRITC-conjugated avidin (Pierce) andFITC-conjugated secondary antibodies, and were observed under theOlympus Fluoview laser scanning confocal imaging system (Olympus Inc.).

Co-localization between ES and cell surface can also be observed invivo. The biotinylated ES and antibodies against NL were injectedintravenously into mice bearing B16-F10 tumors. The biotinylated ES andpurified rabbit IgG was injected i.v. as a control. Tissues werecollected 1 h after injection, and immunofluorescence was performed. Thebiotin-labeled ES and antibodies against NL selectively accumulated onthe surface of tumor blood vessels (FIG. 7 j-l). None of them could bedetected on the blood vessels of other tissues such as heart (FIG. 7a-c), liver (FIG. 7 d-f), and kidney (FIG. 7 g-i). In tumor tissue, aperfect merge between biotinylated ES and antibodies against NL wasobserved. Control IgG was not detectable in the tumor tissues (FIG. 7m-o). These results suggest that ES and NL are co-localized on thesurface of tumor blood vessels but not blood vessels of other normaltissues.

All papers, publications, literature, patents, patent applications,websites, and other printed or electronic documents referred herein,including but not limited to the references listed below, areincorporated by reference in their entirety.

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1. A kit for determining the susceptibility of a subject to endostatincancer therapy, comprising: a. A label that labels nucleolin; and b. Ausage instruction for performing a screening of a sample of said subjectwith said label such as that an amount of nucleolin present in thesample is determined.
 2. The kit of claim 1, wherein the subject is amammal.
 3. The kit of claim 1, wherein the subject is a human.
 4. Thekit of claim 1, wherein the nucleolin being labeled is cell surfacenucleolin.
 5. The kit of claim 1, wherein the nucleolin being labeled issystemic nucleolin.
 6. The kit of claim 1, wherein the label comprisesan antibody that specifically binds to nucleolin.
 7. The kit of claim 6,wherein the antibody is a monoclonal antibody.
 8. The kit of claim 6,wherein the antibody is a polyclonal antibody.
 9. The kit of claim 1,wherein the label comprises a nucleic acid molecule.
 10. The kit ofclaim 9, wherein the nucleic acid molecule is a DNA molecule.
 11. Thekit of claim 9, wherein the nucleic acid molecule is an RNA molecule.12. A method of determining the likelihood of success of endostatincancer therapy in a subject, comprising: a. Screening a sample from saidsubject for the level of expression of nucleolin; and b. Determining ifsaid subject is susceptible to endostatin cancer therapy based on theamount of nucleolin expression.
 13. A method of producing anucleolin-specific angiogenesis inhibitor effective in inhibitingangiogenesis, comprising: a. Applying an appropriate binding assay to apool of candidate molecules, thereby obtaining a plurality ofnucleolin-specific molecules; b. Testing each of the plurality ofnucleolin-specific molecules for its effectiveness of inhibitingangiogenesis using an anti-angiogenesis assay; and c. Selecting theresulting nucleolin-specific molecule which is effective in inhibitingangiogenesis as demonstrated by the anti-angiogenesis assay.
 14. Themethod of claim 13, wherein the nucleolin-specific angiogenesisinhibitor is a protein or peptide.
 15. The method of claim 13, whereinthe nucleolin-specific angiogenesis inhibitor is a small molecule. 16.The method of claim 13, wherein the nucleolin-specific angiogenesisinhibitor is used in curing an angiogenesis-dependent disease.
 17. Themethod of claim 16, wherein the angiogenesis-dependent disease is acancer.
 18. The method of claim 16, wherein the angiogenesis-dependentdisease is an endothelial cell disease.
 19. A method of selecting anangiogenesis inhibitor having the ability to inhibit endothelialproliferation and/or migration when added to proliferating endothelialcells in vitro, comprising the steps of: a. Using a pharmaceuticallyacceptable method to discover molecules that specifically interact withnucleolin as the target molecule; b. Testing the molecules thus derivedfrom step (a) for their effectiveness in inhibiting endothelial cellproliferation and/or migration; and c. Determining the molecule thusderived which are effective in inhibition of endothelial cellproliferation and/or migration, wherein the effectiveness of theanti-angiogenesis function of said molecule is compared to that ofendostatin.
 20. The method of claim 19, wherein the nucleolin-specificmolecule is a protein or polypeptide.
 21. The method of claim 19,wherein the nucleolin-specific molecule is a small molecule.
 22. Amethod of increasing the receptiveness of a target cell to anangiogenesis inhibitor, comprising: a. Introducing an exogenousnucleolin into the target cells, thereby obtaining a plurality ofmodified target cells expressing the exogenous nucleolin; and b.Measuring the killing rate of the modified target cells by endostatin.23. The method of claim 22, wherein the target cell is a cancer cell.24. The method of claim 22, wherein the target cell is an endothelialcell.
 25. The method of claim 22, wherein said angiogenesis inhibitor isendostatin.
 26. The method of claim 22, wherein the exogenous nucleolinis introduced into the target cell via a viral vector.
 27. A method ofenhancing the anti-angiogenesis effect of an angiogenesis inhibitor on atarget endothelial cell, comprising a. Introducing into said target cella pharmaceutically effective amount of exogenous nucleolin molecule,said nucleolin molecule being able to express in said target cell; andb. Incubating said target cell with said angiogenesis inhibitor, therebycausing the inhibition of the growth of said target cell.
 28. The methodof claim 27, wherein said angiogenesis inhibitor is endostatin.
 29. Themethod of claim 27, wherein said target endothelial cell is a cancercell.
 30. A diagnostic kit for assaying the individual sensitivity oftarget cells towards angiogenesis inhibitors, comprising: a. A moleculethat specifically bind to an nucleolin molecule; and b. Apharmaceutically acceptable carrier.
 31. The diagnostic kit of claim 30,wherein the angiogenesis inhibitor is endostatin.
 32. The diagnostic kitof claim 30, wherein the target cell is a cancer cell.
 33. A diagnostickit for determining the target cancer cells susceptible toanti-angiogenesis inhibitor treatment, comprising: a. An antibodyagainst nucleolin; and b. A pharmaceutically acceptable carrier.
 34. Thediagnostic kit of claim 33, wherein the antibody is a polyclonalantibody.
 35. The diagnostic kit of claim 33, wherein the antibody is amonoclonal antibody.
 36. A method of determining a patient's sensitivityto endostatin therapy, comprising contacting a sample of a patient withan antibody against nucleolin, and detecting the formation of a complexbetween the antibody and nucleolin, a higher level of presence of thecomplex indicating a higher likelihood of success of endostatin therapy.37. A method of increasing the efficacy of angiogenesis inhibitors oncontrolling the growth of a cancer in a patient having such cancer,comprising: a. Identifying the presence of the level of endogenousnucleolin molecules in a sample of the cancer of said patient; and b.Determining the likelihood of efficacy of angiogenesis inhibitor on saidcancer patient using the level of expression of nucleolin in saidpatient, a higher level of nucleolin indicating a higher degree ofsuccess of such angiogenesis inhibitor treatment.
 38. The method ofclaim 37, wherein said angiogenesis inhibitor is endostatin.
 39. Themethod of claim 37, wherein the detection of nucleolin level in suchsample of said patient is performed by immunoprecipitation ofanti-nucleolin antibody and nucleolin in said sample.
 40. A method ofidentifying target cancer cells which are susceptible to ananti-angiogenesis inhibitor treatment, comprising: a. Generating ananti-nucleolin antibody; b. Contacting a sample from a subject with saidanti-nucleolin antibody; and c. Identifying target cancer cells that aresusceptible to anti-angiogenesis inhibitor treatment as indicated by thelevel of nucleolin present in the sample, a higher level indicating ahigher susceptibility.
 41. The method of claim 40, wherein theanti-nucleolin antibody is polyclonal antibody.
 42. The method of claim40, wherein the anti-nucleolin antibody is monoclonal antibody.
 43. Themethod of claim 40, wherein the anti-angiogenesis inhibitor isendostatin.
 44. A method of inhibiting the proliferation and/ormigration of a plurality of endothelial cells in a cell sample,comprising: a. Linking an anti-nucleolin antibody with a cytotoxicagent, thereby forming an anti-nucleolin toxic antibody; and b.Administering said anti-nucleolin toxic antibody to said cell sample,thereby inhibiting the proliferation and/or growth of said plurality ofendothelia cells.
 45. The method of claim 44, wherein the cytotoxicagent is a cytokine.
 46. The method of claim 44, wherein the cytotoxicagent is a tumor necrosis factor.
 47. The method of claim 44, whereinthe cell sample is derived from cancer patients.